First Report of Grapevine Pinot gris virus Infecting Grapevine in Slovenia.

Unusual virus-like symptoms were first observed in 2001 on grapevine cvs. Pinot gris and Sauvignonasse in vineyards from the western part of Slovenia. Symptomatic plants showed shortened internodes, poor leaf development, mottling, and deformations of leaves that resulted in poor growth of symptomatic plants. In 2003 and 2004, several samples were tested for Arabis mosaic virus, Cherry leafroll virus, Grapevine fanleaf virus, Raspberry bushy dwarf virus, Strawberry latent ringspot virus, Tomato black ring virus, Tomato ringspot virus, and Tobacco ringspot virus by DAS-ELISA, but none of them could be confirmed as the cause of the observed symptoms. During intensive visual inspections between 2002 and 2006, the symptoms were observed on most grapevine cultivars grown in the Primorska region but predominantly on the two previously mentioned cultivars. In Trentino, northern Italy, similar virus-like symptoms, i.e., chlorotic mottling, puckering and deformation of the leaves, reduced yield, and low quality of the berries were observed in grapevine plants cv. Pinot gris in 2003 and in cvs. Traminer and Pinot noir in 2009 (2). No common grapevine viruses could be associated with the disease. In 2012, a new trichovirus named Grapevine Pinot gris virus (GPGV) was found in Pinot gris plants using deep sequencing. The virus was also detected in symptomless plants (2). GPGV was later reported also from Korea causing inner necrosis of berries and poor fruit set in grapevine cv. Tamnara (1). In 2012, 42 leaf samples from mostly symptomatic grapevine plants of cvs. Pinot gris, Pinot noir, and Muscat blanc were collected at three locations in the Primorska region. Total RNA was extracted from leaves using the MagMAX Express magnetic particle processor with MagMAX-96 Total RNA Isolation Kit and Plant RNA Isolation Aid in Lysis Binding Solution Concentrate (all by Life Technologies, Grand Island, NY). DNA fragments of 1,049 bp corresponding to the movement protein gene were successfully amplified by RT-PCR from 40 samples using primers GPgV5619 and GPgV6668 (2). Amplification products from three plants were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and sequenced. The sequences were deposited in the EBI database under the accession numbers HG738850 to 52. All the nucleotide sequences shared 97.4 to 97.6% identity with GPGV from Italy (sequence FR877530) and 97.1 to 98.2% amino acid identity within the translated region. To our knowledge, this is the first report of GPGV in Slovenia. The disease seems to be spreading extensively in the Primorska region, causing considerable economic losses, and in 2013 it was also observed in other regions of Slovenia. Since the virus could be found in symptomless plants in Italy and in Slovenia, its role in the development of the disease should be further investigated. References: (1) I. S. Cho et al. New Dis. Rep. 27:10, 2013. (2) A. Giampetruzzi et al. Virus Res. 163:262, 2012.

First Report of Pseudomonas syringae pv. actinidiae, the Causal Agent of Bacterial Canker of Kiwifruit in Slovenia.

In May 2013, 20 plants in a production orchard of kiwifruit (Actinidia deliciosa) cv. Hayward in the seaside area of Primorska showed small, angular, coalescing necrotic leaf spots and cankers on green shoots. In the following 2 weeks, disease progressed to wilting and shoot dieback with exudates. Symptoms were consistent with Pseudomonas syringae pv. actinidiae. Circular, flat, granulated colonies with entire margins were isolated from leaf spots on King's medium B (KB) and on sucrose nutrient agar with boric acid, cephalexine, and cycloheximide. Strains were purified on KB and showed weak fluorescence upon a prolonged incubation (>10 days) and belonged to P. syringae LOPAT group Ia (+---+). DNA was extracted from strains and plant extracts with Chelex 100 resin and Bio-Nobile QuickPick Plant Kit (Turku, Finland), respectively. PCR products of expected sizes were generated by PCR assays (2,4) from all strains and plant extract, supporting the strains as being P. syringae pv. actinidiae. Two strains (NIB Z 1870 and 1871) were further identified by cytochrome C oxidase (negative), glucose metabolism (oxidative), aesculine (negative), and nitrate (negative). Their partial rpoD gene sequences (GenBank Accession Nos. KJ724117 and KJ724118) (3) were identical to the sequence of the P. syringae pv. actinidiae pathotype strain NCPPB 3739 (FN433222, 100% coverage) and to the sequence of P. syringae pv. theae at 96% coverage (FN433271). BOX-PCR fingerprinting and multilocus sequence analysis (MLSA) based on four housekeeping genes gapA (KJ733923 and KJ733924), gltA (KJ733925 and KJ733926), gyrB (KJ733927 and KJ733928), and rpoD identified both strains as biovar 3, a highly virulent biovar of P. syringae pv. actinidiae (5). The pathogenicity of the two strains was confirmed on four plants of A. deliciosa 'Hayward' for each strain. Six-month-old plants were sprayed on the abaxial sides of leaves with 30 ml cell suspension prepared from a 72-h-old culture of the appropriate strain (~8 × 106 CFU/ml in 0.01 M MgSO4), covered with plastic bags for 24 h, and incubated under high relative humidity (80%) with 14 h daylight and 24/21°C day/night temperature. Three positive and three negative control plants were inoculated with the Italian P. syringae pv. actinidiae virulent strain K9 (kindly provided by Dr. Gian Luca Bianchi of the Plant Health Service of Friuli Venezia Giulia region) and 0.01 M MgSO4, respectively. After 7 days, water-soaked brown spots with pale green halos were observed on all plants inoculated with bacteria. Re-isolated bacteria were identical to the original strains in their morphology, PCR products, and rpoD sequences. Negative control plants did not develop symptoms, and no growth was observed on media. This is the first laboratory confirmation of bacterial canker of kiwifruit in Slovenia. Visual inspections carried out by the plant health authorities in 2013 and laboratory analysis confirmed additional infection with P. syringae pv. actinidiae in a single, nearby orchard. The pest status of P. syringae pv. actinidiae in Slovenia is officially declared as present, subject to official control (1). References: (1) EPPO Reporting Service. Online publication: http://archives.eppo.int/EPPOReporting/2014/Rse-1402.pdf . No. 02 2014/026, 2014. (2) A. Gallelli et al. J. Plant Pathol 93:425, 2011. (3) N. Parkinson et al. Plant Pathol. 60:338, 2011. (4) J. Rees-George et al. Plant Pathol. 59:453, 2010. (5) J. L. Vanneste et al. Plant Dis. 97:708, 2013.

First Report of Raspberry bushy dwarf virus on Red Raspberry and Grapevine in Slovenia.

In 2002, severe vein yellowing and partial or complete yellowing of leaves was observed on some shoots of red raspberry (Rubus idaeus) cvs. Golden Bliss and Autumn Bliss. Sap of infected plants of cv. Golden Bliss was inoculated onto Chenopodium quinoa and Nicotiana benthamiana. Faint chlorotic spots were observed on inoculated leaves of C. quinoa approximately 14 days after inoculation but no systemic symptoms appeared. No symptoms were observed on N. benthamiana. Raspberry bushy dwarf virus (RBDV) was detected in the original raspberry plant using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antiserum (Loewe Biochemica, Sauerlach, Germany). Systemic infections of inoculated C. quinoa and N. benthaminana were confirmed using DAS-ELISA. In 2001 and 2002, unusual virus symptoms were observed on grapevine grafts (Vitis vinifera) of cv. Laski Rizling. Symptoms appeared as curved line patterns and yellowing of the leaves. No nepoviruses were found in symptomatic plants, but RBDV was confirmed using DAS-ELISA. RBDV infection was later confirmed in grapevine cv. Stajerska Belina with similar symptoms. RBDV was transmitted mechanically from grapevine to C. quinoa where it was detected by immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR). IC-RT-PCR was used to amplify a part of the coat protein gene of the virus from raspberry and grapevine, and the amplification products were sequenced (1). The obtained sequence shared at least 93% nucleotide sequence identity with other known RBDV sequences, which confirmed the serological results. To our knowledge, this is the first report of the natural occurrence of RBDV in grapevine and also of RBDV infection of red raspberry in Slovenia. Reference: (1) H. I. Kokko et al. Biotechniques 20:842, 1996.

Testing the efficacy of different insecticides to control onion thrips (Thrips tabaci Lindeman, Thysanoptera, Thripidae) in onion crops.

Onion thrips (Thrips tabaci) has become important pest in onion crops in Slovenia. Because it is a polyphagous species, it causes damage also in vegetable production in general (leek, cabbage, ornamentals etc.). The group of efficient insecticides for thrips control in Slovenia is limited, because its misusage in the past caused the development of resistance of this pest. In 2002, eight different insecticides based on four active substances was tested in Goriska region. The results of field experiments showed the highest efficiency of active substances Spinosad and Abamectine for control of Thrips tabaci. Unfortunatelly, they are not registrated in Slovenia for this purpose as yet.